Axon injury is an early event of neurotrauma that leads to retrograde cellular changes, including somatic ER stress, synapse loss, hyper-excitability, and even cell death. Foundational work remains needed to understand how axon-to-soma injury signals propagate to effect these profound retrograde changes in long projection pyramidal cells. Understanding this signaling is critical for the future development of neuroprotective approaches. Axon injury causes massive influx of calcium into the cytosol to initiate axon-to-soma signaling, leading to transcription-dependent retrograde synapse loss and hyperexcitability. Evidence suggests either an endoplasmic reticulum (ER)-dependent calcium wave or calcium-primed microtubule-based transport mediates this long-range axon-to-soma signaling. A vast ER network extends throughout the neuron from distal axon terminals to dendritic spines, influencing the availability of calcium, but the extent of axonal ER involvement in axon injury-induced synapse loss and hyper-excitability remains unknown. Further, while rat injury models are a mainstay of this research, it remains unclear how human glutamatergic neurons, with their diminished regenerative capacity, differ in their injury signaling mechanisms. Experimentally tractable multi-compartment, microfluidic chambers enable manipulation of axons independently from somata and dendrites, providing an important tool to investigate axon-to-soma communication in both murine and human stem cell-derived neurons. We found that hippocampal pyramidal neurons subjected to distal axotomy in our microfluidic chambers undergo somatic ER stress, retrograde synapse loss, and hyper-excitability. Reducing calcium influx locally at the site of injury and blocking transcription prevents axotomy-induced dendritic spine loss; thus, calcium signaling and rapid transcription mediate synapse loss following axon injury. Our long-term goal is to identify key molecular players and their timing of action that cause retrograde synapse loss and hyper-excitability following axon injury. Aim 1 will determine the influence of axonal ER on axotomy-induced somatic ER stress in both rat and human glutamatergic neurons. Aim 2 will examine the influence of axonal ER signaling on axotomy-induced synapse loss and hyper-excitability. Together, this study provides a critical first step in defining the role of ER during axon-to-soma injury signaling in pyramidal cells. Further, this project may lead to the novel identification of therapeutics during early stages of neuron damage and will likely have broader implications for other neurological disorders where both ER stress and axon damage are prevalent.